Dendritic spines are the primary postsynaptic targets for excitatory glutamatergic synapses in the brain. They are highly dynamic structures that undergo changes in size, shape, and number during development, as well as in response to physiological stimuli such as learning. Spine development involves three processes: formation, maturation, and pruning. Pruning appears to be an activity-dependent process and likely plays a significant role in the refinement of synaptic connections. While many proteins have been found to control spine formation and early spine maturation, very little is known about the molecular mechanisms that mediate the late phase of spine maturation and pruning. Therefore, understanding the mechanisms regulating spine morphogenesis will provide significant insight into processes fundamental for brain development and synaptic plasticity, as well as the pathology of some neurological diseases. Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic plasticity, particularly at glutamatergic synapses containing AMPA receptors. The gene for BDNF produces two pools of mRNA, with either a short or long 3'untranslated region (3'UTR). Previous studies from my lab show that short 3'UTR Bdnf mRNA is restricted to the soma, while long 3'UTR Bdnf mRNA is present in both the soma as well as dendrites for local translation. The overall objective of this research proposal is to understand the molecular mechanisms through which BDNF regulates dendritic spine morphogenesis and synapse regulation. Specifically, the proposed experiments plan to dissociate the roles of somatically and dendritically synthesized BDNF in the regulation of glutamatergic synapses (Aim1). I propose to use an in vitro assay I have recently developed for the study of spine morphogenesis, which mimics the in vivo course of spine development in the rodent hippocampus. In this assay, actin-GFP-labeled spines of cultured rat hippocampal neurons form during the first 2 weeks, mature during weeks 3 and 4, and are pruned during week 4. Using immunocytochemistry, NBQX and whole-cell patch-clamp recordings, I will test my hypothesis that somatically and dendritically synthesized BDNF exert opposing effects on AMPA-type glutamate receptor composition, function and signaling. In addition to this set of experiments, I plan to elucidate the signaling pathway by which dendritically synthesized BDNF regulates spine pruning (Aim 2). I hypothesize that activity induces dendritic translation and secretion of proBDNF, which interacts with p75[NTR] to mediate spine pruning through RhoA in cultured hippocampal neurons. To test my hypothesis, I will employ adeno-associated viruses, shRNA constructs, p75[NTR] KO mice, whole-cell patch-clamp recordings, GST-pull down assay, Western blot and a cleavage-resistant proBDNF construct I have recently generated. Taken together, these experiments will demonstrate distinct roles of somatically and dendritically synthesized BDNF in spine morphogenesis and may identify a signaling cascade through which dendritically synthesized BDNF regulates spine pruning in cultured hippocampal neurons.